Our goal is to develop a first -in-class therapeutic agent that directly blocks the metabolism of fructose, a key component in sugar. Intake of sugar (sucrose) and high fructose corn syrup (HFCS) induces metabolic syndrome and diabetes in laboratory animals and are strongly associated with obesity and diabetes in humans. Both sucrose and HFCS contain fructose, which stimulates food intake by inducing leptin resistance while lowering metabolism, including blocking fatty-acid oxidation. We found that these effects of fructose were mediated by the unique ability of fructose to decrease intracellular-ATP levels during its metabolism, which is due to the rapid consumption of ATP by the enzyme fructokinase C (KHK-C) in the liver. Mice lacking KHK-C are protected from sugar-induced obesity, fatty liver, and metabolic syndrome. While sugar and HFCS are major sources of dietary fructose, we found that fructose can be endogenously produced by high glycemic or high salt diets and that blocking fructokinase protects animals from obesity and insulin resistance from this source of fructose. Furthermore, mice lacking fructokinase are also protected from the orphan disease, hereditary fructose intolerance (HFI). Blocking KHK-C is safe, as humans lacking have a normal life span and mice lacking KHK-C are even protected from aging-associated kidney disease. In Phase I of our STTR, our goal was to develop two novel lead compounds with IC50 values in the submicromolar range that were effective at blocking KHK-C both in vitro and in vivo and with specificity for KHK compared to other sugar kinases. While much of our initial effort was aimed at developing derivatives of a nutraceutical (osthol) as novel chemical entities, we were unable to break the submicromolar-IC50 target. However, more recently a series of novel indazole compounds has been identified, which have high potency and selectivity, and have robust in vivo activity after oral administration. In this Phase II proposal, we plan to optimize our lead inhibitors using structure-based drug design (SBDD). Aim 1 is to optimize our lead inhibitors with increased potency (IC50 <40 nM) and selectivity guided by SBDD. In Aim 2, the most promising lead compounds will be evaluated in in vitro ADME assays (e.g. solubility, permeability, microsomal stability) and in vitro toxicity (e.g. CYP450 inhibition, Ames test and hERG inhibition. Inhibitors of high interest will undergo in vivo pharmacokinetic (PK) profiling and exploratory toxicity testing. Aim 3 is to test these lead compounds in our unique animal disease models for long-term efficacy at blocking fructose-induced fatty liver, nonalcoholic fatty liver disease, and metabolic syndrome, as well the protection of mice with HFI (due to knockout of the aldolase B gene) towards fructose. Inhibitors with robust efficacy will also be evaluated in exploratory toxicology studies. Completion of these studies should result in an optimal preclinical development candidate for a first-in- class drug for those suffering from HFI and metabolic syndrome.